Spatiotemporally Controlled T‐Cell Combination Therapy for Solid Tumor

Abstract Due to multidimensional complexity of solid tumor, development of rational T‐cell combinations and corresponding formulations is still challenging. Herein, a triple combination of T cells are developed with Indoleamine 2,3‐dioxygenase inhibitors (IDOi) and Cyclin‐dependent kinase 4/6 inhibitors (CDK4/6i). To maximize synergism, a spatiotemporally controlled T‐cell engineering technology to formulate triple drugs into one cell therapeutic, is established. Specifically, a sequentially responsive core‐shell nanoparticle (SRN) encapsulating IDOi and CDK4/6i is anchored onto T cells. The yielded SRN‐T cells migrated into solid tumor, and achieved a 1st release of IDOi in acidic tumor microenvironment (TME). Released IDOi restored tryptophan supply in TME, which activated effector T cells and inhibited Tregs. Meanwhile, 1st released core is internalized by tumor cells and degraded by glutathione (GSH), to realize a 2nd release of CDK4/6i, which induced up‐regulated expression of C‐X‐C motif chemokine ligand 10 (CXCL10) and C‐C motif chemokine ligand 5 (CCL5), and thus significantly increased tumor infiltration of T cells. Together, with an enhanced recruitment and activation, T cells significantly suppressed tumor growth, and prolonged survival of tumor‐bearing mice. This study demonstrated rationality and superiority of a tri‐drug combination mediated by spatiotemporally controlled cell‐engineering technology, which provides a new treatment regimen for solid tumor.


Chemistry
To prepare GSH responsive liposomal core, lipidic CTA were designed and synthesized in this paper.Lipidic CTA is composed of hydrophobic tail chain double-stranded tetrahexanol, intermediate arm disulfide bond, and trithiol bond in the head.The hydrophobic tail chain can participate in the formation of the bimolecular skeleton of liposomes.The disulfide bond can respond to the high concentration of GSH in the cytoplasmic rupture to release drugs, and the trithiol bond can be used as a chain transfer agent to initiate the PET-RAFT polymerization reaction.These structures were confirmed by 1 H-NMR, MS or HRMS.
Then, n-tetradecanol (5.0 g, 23.3 mmol) was added and refluxed for 12 h.Then, the solvent toluene was removed by vacuum distillation.Recrystallize the concentrated solution with 30 mL of methanol to obtain a white solid.This solid was dissolved in dichloromethane and washed with sodium bicarbonate aqueous solution (5%, 15 mL×2), and saturated salt water in sequence.
Subsequently, the organic layer was separated, and dried over anhydrous Na2SO4 and evaporated.
The pH of the water layer was adjusted to 9.0 using a 1M NaOH aqueous solution, and then extracted with ethyl acetate (40 mL×2).The organic layer was separated, and dried over anhydrous Na2SO4 and evaporated.The residue was obtained to give Compound 2 as a white oily solid (1.57g, 46.8%).MS, ESI + , m/z: Calcd for C9H20N2O2S2 (M+H) + , 252.10; Found, 253.10.

Isolation of mouse CD8 + T cells
Spleens from C57BL/6J Thy1.1 + pmel-1 mice were ground through a 70-μm filter, and red blood cells were removed by incubation with ACK lysis buffer for 5 min at 4°C.Then, the splenic cells were centrifuged, washed with D-PBS (without Ca 2+ and Mg 2+ ), and isolated by EasySep TM Mouse CD8 + T cell Isolation Kit (StemCell) to obtain naïve pmel-1 Thy1.1 + CD8 + T cells (CD8 + T cells).For activated CD8 + T, naive CD8 + T cells were resuspended at 1.5×10 6 cells per milliliter in RPMI medium containing 10 ng/mL recombinant mouse IL-2, plate-bound 5 μg/mL anti-CD3, and 2 μg/mL anti-CD28 agonist antibodies and incubated at 37°C.After an incubation period of 2 days, dead cells were removed by centrifugation and living cells were collected.Activated CD8 + T cells were cultured in a medium containing IL-2 and anti-CD3 and anti-CD28 agonist antibodies for in vitro and in vivo studies.

Preparation and characterization of SRN
Liposomal core was prepared by using a film dispersion method.Briefly, 50 mg natural soybean phosphatidylcholine (S100), 9 mg cholesterol (Chol), 3.5 mg CDK4/6i and 12 mg lipidic CTA were dissolved in 5 mL of mixture of CHCl3 and MeOH (3: 2,v: v).After the organic solvents were evaporated at 40℃, a thin lipid film was formed.Then the organic solvents were then further removed under vacuum overnight.The lipid film was hydrated with 3.5 mL of the distilled water at 37℃ for 30 min, then the liposomes were obtained by dispersion using an ultrasonic cell disruptor under ice bath (30% power, 15 min) and extrusion through polycarbonate membrane filters with a pore size of 0.45 μm.
Using the PET-RAFT polymerization reaction to constructe SRN.Berifly, PET-RAFT polymerization was carried out in pH 7.4 aqueous solution to build an acid responsive gel layer containing IDOi on the surface of liposomal core as the shell of SRN.Berifly, acrylamide (4 mg, 0.056 mmol), glycerol methacrylate (1.23 mg, 5.396 µmol), and DBCO-PEGA (386 µg, 386 µ mol) were added into 650 µL aqueous solution of liposomal core.The mixture was placed in a 2 mL transparent glass container with a rubber diaphragm.Then, 1% triethanolamine aqueous solution (10.4 µL, v: v), 0.5 mg/mL Eosin Y aqueous solution (15.0 µL) and 1 mg of IDOi were added to the glass container, repectively.Then, the reaction system was thoroughly stirred and mixed.Then, a 27 G, 1/2 needle was connected to a nitrogen (N2) source and was inserted through a rubber spacer to blow N2.Moreover, another 27 G, 1/2 needle was inserted through the spacer to vent for 10 min.Then, the reaction mixture was stirred in a dark at 250 rpm for 10 min, and turn on the 465 nm (10 mW/cm 2 ) light source, which act the polymerize reaction for 30 min and obtain SRN with a shell core structure.The liposomal core and SRN were diluted to a certain concentration to observe the morphology and particle size by TEM and Dynamic light scattering (DLS) analyzer (Brookhaven).
To investigate the stepwise degraded of SRN, SRN was incubated with PBS (pH 7.4), PBS (pH 6.5), and PBS (pH 6.5) containing 10 mM GSH.After incubated at 37℃ for 4 h, SRN solutions were collected and placed in a dialysis bag with molecular weight cut off 8000-12000 Da.Then, the morphology of nanoparticles in each group was observed and photographed by TEM.
Meanwhile, drug loading stability and particle size changes of SRN under different temperature was also evaluated for different time (24, 48, 72 h) using HPLC and DLS.
To investigate the IDOi and CDK4/6i release from SRN under physiological conditions in vitro，normal blood environment (PBS, containing 0.01 mM GSH) were applied as a dialysis medium.Putting a dialysis bag (molecular weight cut-off of 8000-12000 DA) containing 1 mL SRN of 500 μg/mL CDK4/6i into 50 mL solution of the above physiological conditions, respectively, and incubated at 37℃ in a water bath thermostatic shaker.The sample in of each dialysis bag was collected at different time (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 48 h) for HPLC detection after centrifuging to supernatant, and the cumulative release amounts of CDK4/6i and IDOi at different time points were calculated and the release profiles were plotted.

Figure S2 .
Figure S2.Characterization of SRN structures.a.The hydrodynamic sizes of liposomal core and SRN.b.The colorimetric intensity of the colored hydrogels at different wavelengths via the treatment of photoinitiator Eosin Y and photocatalytic conditions.

Figure S3 .
Figure S3.The stability of SRN at different conditions.a. Particle size changes of SRN in different physiological environments.b-e.Drug loading stability of SRN under different temperature.f.Particle size changes of SRN under different temperature.Data were shown as mean ± SEM (n = 3).

Figure S4 .
Figure S4.Optimize the conditions for preparation of SRN-T cells.a, b.In vitro cytotoxicity of different concentration of DPPE-PEG5k-N3 on CD8 + T cells for 24 h.c.The cells were incubated

Figure S6 .
Figure S6.The cell viability and activation level of SRN-T cells.a. Histogram of the cell viability of T cells during the CTLs expanded.b.Histogram of the activation level of SRN-T cells.Data are represented as mean ± SEM; n = 3; n.s, not significant; determined by one-way ANOVA with Tukey's correction in a or unpaired t test in b.

Figure S9 .
Figure S9.Triple independent experiments of Western blot.

Figure S11 .
Figure S11.Representative flow cytometric analysis of the level of MHC-1 expression of B16F10 cells.

Figure S13 .
Figure S13.The tumor sections after administrated SRN-T cells for different time.T cells were labelled with CFSE and SRN were labelled with Rhodamine B. Scale bar, 200 µm.

Figure S15 .
Figure S15.Biosafety analysis of different treatment groups in B16F10 tumor-bearing mice.n = 4 mice/group.Data are represented as mean ± SEM; n.s, not significant; determined by oneway ANOVA with Tukey's correction.

Figure S16 .
Figure S16.The Safety evaluation of heart, liver, spleen, lung, and kidney tissue by microscopic pathological analysis in B16F10 orthotopic melanoma tumor-bearing mice.Tissues were collected from B16F10 tumor-bearing mice after administration of different treatments.Hematoxylin and eosin (H&E) staining was performed on tissue sections for pathological assessment.Scale bar, 200 μm.